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The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76-93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The K m and V max for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min -1 mg -1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We describe a new species of the genus Alippistrongylus (Nematoda: Trichostrongylina) in the small intestine of Delomys dorsalis (Rodentia: Sigmodontinae) found in Itatiaia National Park (Parque Nacional do Itatiaia, PNI), state of Rio de Janeiro, Brazil. The new species can be distinguished between the other two species previously described by the following morphological characteristics: pattern of the rays from the caudal bursa rays in males and the spherical-shaped appendage in the female. Additionally, we provide molecular genetic data from the new species. The discovery of Alippistrongylus itatiaiaensis sp. n. expands our understanding of nematode diversity and underscores the importance of parasite studies in biodiversity conservation. Its occurrence in a preserved area like the PNI emphasises the role of such habitats in maintaining unique ecological interactions.

Dictyocaulus xanthopygus sp. nov. (Nematoda: Trichostrongyloidea) was isolated from the lungs of the Manchurian wapiti in Primorsky kray, Russia. The newly described species exhibits morphological characteristics of Dictyocaulus but is distinct from congeneric species based on morphological (lengths of body and esophagus, distances from the anterior end to nerve ring and to excretory pore, the thickness of the buccal capsule, etc.) and molecular features. High levels of genetic divergence as well as Bayesian phylogenetic analyses based on 18S rRNA nuclear and cox1 mitochondrial genes supported the independence of Dictyocaulus xanthopygus sp. nov. Secondary structures of helix 39 of 18S rRNA were identical, while ES9 adjacent to the helix has a unique conformation for newly described worms. Energy-efficient conformational rearrangements of rRNA secondary structures can be applicable in studies on the pathogenesis, epidemiology, taxonomy and evolutionary biology of parasites. Additionally, bracketed dichotomous keys to six valid species of Dictyocaulus were prepared., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

A large-scale Fecal Egg Count Reduction Test (FECRT) was integrated with ITS-2 rDNA nemabiome metabarcoding to investigate anthelmintic resistance in gastrointestinal nematode (GIN) parasites in western Canadian beef cattle. The study was designed to detect anthelmintic resistance with the low fecal egg counts that typically occur in cattle in northern temperate regions. Two hundred and thirty-four auction market-derived, fall-weaned steer calves coming off pasture were randomized into three groups in feedlot pens: an untreated control group, an injectable ivermectin treatment group, and an injectable ivermectin/oral fenbendazole combination treatment group. Each group was divided into six replicate pens with 13 calves per pen. Individual fecal samples were taken pre-treatment, day 14 post-treatment, and at monthly intervals for six months for strongyle egg counting and metabarcoding. Ivermectin treatment resulted in an 82.4% mean strongyle-type fecal egg count reduction (95% CI 67.8-90.4) at 14 days post-treatment, while the combination treatment was 100% effective, confirming the existence of ivermectin-resistant GIN. Nemabiome metabarcoding of third-stage larvae from coprocultures revealed an increase in the relative abundance of Cooperia oncophora, Cooperia punctata, and Haemonchus placei at 14 days post-ivermectin treatment indicating ivermectin resistance in adult worms. In contrast, Ostertagia ostertagi third-stage larvae were almost completely absent from day 14 coprocultures, indicating that adult worms of this species were not ivermectin resistant. However, there was a recrudescence of O. ostertagi third stage larvae in coprocultures at three to six months post-ivermectin treatment, which indicated ivermectin resistance in hypobiotic larvae. The calves were recruited from the auction market and, therefore, derived from multiple sources in western Canada, suggesting that ivermectin-resistant parasites, including hypobiotic O. ostertagi larvae, are likely widespread in western Canadian beef herds. This work demonstrates the value of integrating ITS-2 rDNA metabarcoding with the FECRT to enhance anthelmintic resistance detection and provide GIN species- and stage-specific information., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2023 Merck Sharp & Dohme LLC., a subsidiary Merck & Co., Inc., The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Gastrointestinal nematodes (GIN) are amongst the most important pathogens of grazing ruminants worldwide, resulting in negative impacts on cattle health and production. The dynamics of infection are driven in large part by the influence of climate and weather on free-living stages on pasture, and computer models have been developed to predict infective larval abundance and guide management strategies. Significant uncertainties around key model parameters limits effective application of these models to GIN in cattle, however, and these parameters are difficult to estimate in natural populations of mixed GIN species. In this paper, recent advances in molecular biology, specifically ITS-2 rDNA 'nemabiome' metabarcoding, are synthesised with a modern population dynamic model, GLOWORM-FL, to overcome this limitation. Experiments under controlled conditions were used to estimate rainfall constraints on migration of infective L3 larvae out of faeces, and their survival in faeces and soil across a temperature gradient, with nemabiome metabarcoding data permitting species-specific estimates for Ostertagia ostertagi and Cooperia oncophora in mixed natural populations. Results showed that L3 of both species survived well in faeces and soil between 0 and 30 °C, and required at least 5 mm of rainfall daily to migrate out of faeces, with the proportion migrating increasing with the amount of rainfall. These estimates were applied within the model using weather and grazing data and use to predict patterns of larval availability on pasture on three commercial beef farms in western Canada. The model performed well overall in predicting the observed seasonal patterns but some discrepancies were evident which should guide further iterative improvements in model development and field methods. The model was also applied to illustrate its use in exploring differences in predicted seasonal transmission patterns in different regions. Such predictive modelling can help inform evidence-based parasite control strategies which are increasingly needed due climate change and drug resistance. The work presented here also illustrates the added value of combining molecular biology and population dynamics to advance predictive understanding of parasite infections., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: John Gilleard reports financial support was provided by Beef Cattle Research Council. Eric Morgan reports financial support was provided by UK Research and Innovation. Hannah Rose Vineer reports financial support was provided by UK Research and Innovation. John Gilleard reports a relationship with Beef Cattle Research Council that includes: funding grants., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Wildlife hosts may serve as reservoirs for strongyles, which can be transmitted to domestic livestock. Therefore, studies evaluating nemabiome compositions in wildlife ruminants are of great use in assessing the possibility of transmission of important nematode pathogens to domestic sheep in Sweden., Methods: First, fecal samples were collected from roe deer (n = 125), fallow deer (n = 106), red deer (n = 18) and mouflon (n = 13) in south central Sweden during the hunting season in 2019. Second, after fecal examination samples were cultured and the larvae were harvested, followed by DNA extractions. Third, all samples were barcoded and processed for sequence analysis on the PacBio platform. Finally, bioinformatic sequence analysis was conducted with DADA2, while species diversity and richness, as well as interactions between the different hosts, were calculated and analyzed in R., Results: Nematode ITS2 sequences were found in 225 of 262 (86%) samples. In total, 31 taxa were identified, among which 26 (86%) to the species level. These were found in different combinations, among which 24 (77%) occurred in roe deer, 19 (61%) in fallow deer, 20 (65%) in red deer and 10 (32%) in mouflon. Five of the species found are known to be associated with livestock (Chabertia ovina, Haemonchus contortus, Oesophagostomum venulosum, Teladorsagia circumcincta and Trichostrongylus axei). However, in the present study the relative abundance and prevalence of most of these species were low. The most striking exception was T. axei, which was relatively abundant in all wildlife hosts. Mostly a wide range of wildlife specific nematodes such as Ostertagia leptospicularis and Spiculopteragia spp. were identified including the invasive nematode Spiculopteragia houdemeri, which was found for the first time in red deer, fallow deer, and mouflon in Sweden. The difference in the number of shared species between mouflon and all cervids (n = 6) was less than among all three cervids (n = 8)., Conclusion: In this study, we investigated the community structure of parasitic intestinal nematodes in four wildlife hosts, and we found that the majority of the parasite species identified were wildlife specific. We also found a new, potentially invasive species not reported before. After comparing the nemabiome of the wildlife hosts in this study with a previous study in sheep from the same geographical region, we conclude that the horizontal transmission potential appears to be relatively low. Still, cross-infections of nematodes between game and sheep cannot be completely ignored., (© 2022. The Author(s).)

Background: The species composition of cattle gastrointestinal nematode (GIN) communities can vary greatly between regions. Despite this, there is remarkably little large-scale surveillance data for cattle GIN species which is due, at least in part, to a lack of scalable diagnostic tools. This lack of regional GIN species-level data represents a major knowledge gap for evidence-based parasite management and assessing the status and impact of factors such as climate change and anthelmintic drug resistance., Methods: This paper presents a large-scale survey of GIN in beef herds across western Canada using ITS-2 rDNA nemabiome metabarcoding. Individual fecal samples were collected from 6 to 20 randomly selected heifers (n = 1665) from each of 85 herds between September 2016 and February 2017 and 10-25 first season calves (n = 824) from each of 42 herds between November 2016 and February 2017., Results: Gastrointestinal nematode communities in heifers and calves were similar in Alberta and Saskatchewan, with Ostertagia ostertagi and Cooperia oncophora being the predominant GIN species in all herds consistent with previous studies. However, in Manitoba, Cooperia punctata was the predominant species overall and the most abundant GIN species in calves from 4/8 beef herds., Conclusions: This study revealed a marked regional heterogeneity of GIN species in grazing beef herds in western Canada. The predominance of C. punctata in Manitoba is unexpected, as although this parasite is often the predominant cattle GIN species in more southerly latitudes, it is generally only a minor component of cattle GIN communities in northern temperate regions. We hypothesize that the unexpected predominance of C. punctata at such a northerly latitude represents a range expansion, likely associated with changes in climate, anthelmintic use, management, and/or animal movement. Whatever the cause, these results are of practical concern since C. punctata is more pathogenic than C. oncophora, the Cooperia species that typically predominates in cooler temperate regions. Finally, this study illustrates the value of ITS-2 rDNA nemabiome metabarcoding as a surveillance tool for ruminant GIN parasites., (© 2022. The Author(s).)

Classic approaches for antemortem identification of gastrointestinal nematodes (GIN) require coproculture of eggs and morphological examination. While adequate for diagnosis, many PCR techniques cannot easily quantify mixed infections without controls and/or standard curves. Herein, we developed a simple and rapid test for differentiating and quantifying mixed infections of GIN using PCR products separated by capillary electrophoresis. Among the cattle GIN, the ITS2 region is sufficiently distinct in length to delineate among the most common infecting genera, Ostertagia ostertagi = 373 bases (b), Haemonchus contortus (placei) = 366b, Cooperia punctata (oncophora) = 376b, Trichostrongylus axei = 372b, and Oesophagostomum radiatum = 357b. Conserved primers were synthesized that span the ITS2 where one primer was fluorescently labeled with 6-FAM. DNAs from infective L3 were PCR amplified then loaded onto an ABI 3130 sequencer adapted for size fragment analysis. Resulting peak amplitudes were both diagnostic and quantitative on a relative basis. As proof of principle, quantification was performed on PCR fragments from mixed species pairs of Ostertagia ostertagi, Cooperia punctata, and Haemonchus contortus and analyzed using Gene Marker V1.85 software. In all cases, linear responses were observed where R 2  > 0.97 and line slopes ranged between 0.90 and 1.1. When tested on eggs from naturally infected animals, the assay showed superior results on two farms when compared to coproculture and morphological identification. Using wildlife-derived samples, results coincided well with deep amplicon sequencing. The assay is adaptable to large-scale studies, does not require comparative PCR controls, and should be compliant with GIN from small ruminant livestock., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Two new species of Viannaia from the intestine of the North American opossums, Didelphis virginiana (Virginia opossum), and Philander opossum (gray four-eyed opossum), are described based on morphological and molecular data, through an integrative taxonomic approach. Maximum likelihood and Bayesian inference analyses for each dataset and the concatenated dataset were performed using a mitochondrial cytochrome c oxidase subunit 1 (COI) gene, and the nuclear ribosomal internal transcribed spacer region (ITS1-5.8S-ITS2). The phylogenetic analyses revealed 2 new species that occur in Mexico, one from the western state of Colima and another from the southern state of Chiapas. Our phylogenetic trees for both molecular markers and concatenated datasets yielded similar topologies with high bootstrap values and posterior probabilities. Viannaia is recovered as a monophyletic group, but the family Viannaiidae appears as non-monophyletic, due to the position of Travassostrongylus scheibelorum, similar to previous studies. Finally, the morphology of Viannaia and Hoineffia is discussed., (© American Society of Parasitologists 2021.)

A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL-MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92 %) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87 % similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the V max was 275 ± 23 nmoles.min -1 . mg -1 protein and the apparent K m for the substrate isocitrate was 0.7 ± 0.01μM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg 2+ ) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90 %. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Among gastrointestinal nematodes, haematophagous strongylids Haemonchus contortus and Ashworthius sidemi belong to the most pathogenic parasites of both domestic and wild ruminants. Correct identification of parasitic taxa is of crucial importance in many areas of parasite research, including monitoring of occurrence, epidemiological studies, or testing of effectiveness of therapy. In this study, we identified H. contortus and A. sidemi in a broad range of ruminant hosts that occur in the Czech Republic using morphological/morphometric and molecular approaches. As an advanced molecular method, we employed qPCR followed by High Resolution Melting analysis, specifically targeting the internal transcribed spacer 1 (ITS-1) sequence to distinguish the two nematode species. We demonstrate that High Resolution Melting curves allow for taxonomic affiliation, making it a convenient, rapid, and reliable identification tool.

Background: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta., Methods: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L., Results: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT., Conclusions: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

A 615 bp full length cDNA encoding a Teladorsagia circumcincta glutathione transferase (TcGST) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. The predicted protein consisted of 205 amino acids and was present as a single band of about 24 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TcGST with homologues from other helminths showed that the highest identity of 53-68% with haem-binding nematode proteins designated as members of the nu class of GSTs. Substrate binding sites and conserved regions were identified and were generally conserved. The predicted 3-dimensional structures of TcGST and HcGST revealed highly open binding cavities typical of this class of GST, considered to allow greater accessibility to diverse ligands compared with other classes of GST. At 25 °C, the optimum pH for TcGST activity was pH 7, the V max was 1535 ± 33 nmoles.min -1 . mg -1 protein and the apparent K m for the substrate 1-chloro-2,4-dinitrobenzene (CDNB) was 0.22 ± 0.01 mM (mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep, recognised recombinant TcGST in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins. These findings could aid in the design of novel drugs and vaccine antigens for economically important parasites of livestock., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 β-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field.

Background: Infections of small ruminants with trichostrongyloid nematodes often result in reduced productivity and may be detrimental to the host. Anthelmintic resistance (AR) against most anthelmintic drug classes is now widespread amongst the trichostrongyloids. Baseline establishment, followed by regular monitoring of the level of AR, is necessary for farmers and veterinarians to make informed decisions about parasite management. The detection of single nucleotide polymorphisms (SNPs) is a sensitive method to detect AR against benzimidazoles (BZs), one of the most widely used anthelmintic classes. Alpine transhumance constitutes a special type of pasturing of sheep from many different farms, the aim of this study was to investigate the prevalence of benzimidazole resistance alleles in this particular management system., Results: Sixteen sheep flocks in Styria and Salzburg in Austria were examined by pyrosequencing for SNPs at codons 167, 198 and 200 of the isotype-1 β-tubulin gene. The frequency of the resistance-associated exchange F200Y was 87-100% for H. contortus, 77-100% for T. colubriformis and <  5-66% for T. circumcincta. Additionally, the F167Y polymorphism was detected in T. colubriformis from two farms at a frequency of 19 and 23% respectively., Conclusions: The high resistance allele frequency in H. contortus and T. colubriformis in the examined sheep population urgently calls for the development of new treatment strategies to sustainably control trichostrongyloid infections for this kind of pasturing, since the frequent mixing of flocks during the alpine summer grazing must be considered an important risk factor for the spread of resistant nematodes to a large number of farms.

Cooperia spp. are parasitic nematodes parasitizing in small intestine of ruminants with a worldwide distribution. Infection of ruminants with Cooperia species can cause severe enteritis, causing significant socio-economic losses to the livestock industry. However, it is yet to know whether there is genetic diversity in mitochondrial (mt) DNA sequences of Cooperia nematodes from different geographic regions. The objective of the present study was to examine sequence difference in mt genomes between Cooperia sp. from China and other Cooperia species. We determined the sequences of the internal transcribed spacer (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of 11 Cooperia specimens collected from the small intestine of a Tianzhu White yak in Gansu Province, northwestern China, which had 99% similarity with that of C. oncophora from Brazil (GenBank accession Number: AJ544290) in ITS-1, and 99% similarity with those from Denmark (AB245040), Scotland and Australia (AJ000032) in ITS-2, indicating that specimens used in the present study should at least represent parasites in Cooperia. We then determined the complete mt genome sequences of one representative specimen of Cooperia sp. from China (CspC), compared the mt DNA sequences with that of C. oncophora from Australia (COA, GQ888713), and conducted phylogenetic analysis with selected nematodes using both maximum likelihood (ML) and Bayesian inference (BI) methods based on both concatenated 12 PCGs, rrnL and rrnS sequences and partial cox2 sequences. The complete mt genome sequence of CspC (KY769271) is 13, 583 bp in length, which is 91 bp shorter than that from COA. The sequence difference over the entire mt genome between CspC and COA was 12.2% in nucleotide and 6.3% in inferred amino acids, with nad4L and nad1 being the most variable and the most conserved PCGs, respectively. Phylogenetic analysis indicated that CspC and COA were closely-related but distinct taxa. The determination of mt genome sequences for Cooperia sp. from China also provides novel resources for further studies of taxonomy, systematics and population genetics of Cooperia from different geographical locations., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms., Methods: Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 β-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur., Results: Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5-90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC 50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples., Conclusions: To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.

Standard diagnostic methods currently in use for the identification of helminth infections in ruminants are based on the morphological analysis of immature and adult stages of parasites. This paper describes a method for the semiquantitative identification of nematodes, mainly Trichostrongyloidea, at species-level resolution. The method is based on amplification and fragment analysis followed by minisequencing of the ITS-2 region (internal transcribed spacer 2) of the ribosomal DNA of parasite eggs or larvae. This method allows for the identification of seven genera (Chabertia, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, Teladorsagia, and Trichostrongylus) and 12 species (Chabertia ovina, Cooperia curticei, Cooperia punctata, Cooperia oncophora/Cooperia surnabada, Haemonchus contortus, Haemonchus placei, Haemonchus longistipes, Oesophagostomum asperum, Oesophagostomum radiatum, Ostertagia ostertagi, Trichostrongylus axei, and Trichostrongylus colubriformis) of infectious nematodes of domestic ruminants. The concordance between the morphological and molecular analyses in the detection of genera ranged from 0.84 to 0.99, suggesting the proposed detection method is specific, semiquantitative, less laborious, and highly cost-efficient.

Background: Parasitic trichostrongyloid nematodes have a worldwide distribution in ruminants and frequently have been reported from humans in Middle and Far East, particularly in rural communities with poor personal hygiene and close cohabitation with herbivorous animals. Different species of the genus Trichostrongylus are the most common trichostrongyloids in humans in endemic areas. Also, Ostertagia species are gastrointestinal nematodes that mainly infect cattle, sheep and goats and in rare occasion humans. The aim of the present study was to identify the trichostrongyloid nematodes obtained from a familial infection in Guilan province, northern Iran, using morphological and molecular criteria., Methods: After anthelmintic treatment, all fecal materials of the patients were collected up to 48 h and male adult worms were isolated. Morphological identification of the adult worms was performed using valid nematode keys. Genomic DNA was extracted from one male worm of each species. PCR amplification of ITS2-rDNA region was carried out, and products were sequenced. Phylogenetic analysis of the nucleotide sequence data was performed using MEGA 6.0 software., Results: Adult worms expelled from the patients were identified as T. colubriformis, T. vitrinus and Teladorsagia circumcincta based on morphological characteristics of the males. Phylogenetic analysis illustrated that each species obtained in current study was placed together with reference sequences submitted to GenBank database., Conclusions: The finding of current study confirms the zoonotic aspect of Trichostrongylus species and T. circumcincta in inhabitants of Guilan province. The occurrence of natural human infection by T. circumcincta is reported for the first time in Iran and the second time in the world.

Recent reports of monepantel (MPTL) resistance in UK field isolates of Teladorsagia circumcincta has highlighted the need for a better understanding of the mechanism of MPTL-resistance in order to preserve its anthelmintic efficacy in this economically important species. Nine discrete populations of T. circumcincta were genotypically characterised; three MPTL-susceptible isolates, three experimentally selected MPTL-resistant strains and three field derived populations. Full-length Tci-mptl-1 gene sequences were generated and comparisons between the MPTL-susceptible isolates, MPTL-resistant strains and one field isolate, showed that different putative MPTL-resistance conferring mutations were present in different resistant isolates. Truncated forms of the Tci-mptl-1 gene were also observed. The genetic variability of individual larvae, within and between populations, was examined using microsatellite analyses at 10 'neutral' loci (presumed to be unaffected by MPTL). Results confirmed that there was little background genetic variation between the populations, global F ST <0.038. Polymorphisms present in exons 7 and 8 of Tci-mptl-1 enabled genotyping of individual larvae. A reduction in the number of genotypes was observed in all MPTL-resistant strains compared to the MPTL-susceptible strains that they were derived from, suggesting there was purifying selection at Tci-mptl-1 as a result of MPTL-treatment. The potential link between benzimidazole (BZ)-resistance and MPTL-resistance was examined by screening individual larvae for the presence of three SNPs associated with BZ-resistance in the β-tubulin isotype-1 gene. The majority of larvae were BZ-susceptible homozygotes at positions 167 and 198. Increased heterozygosity at position 200 was observed in the MPTL-resistant strains compared to their respective MPTL-susceptible population. There was no decrease in the occurrence of BZ-resistant genotypes in larvae from each population. These differences, in light of the purifying selection at this locus in all MPTL-resistant isolates, suggests that Tci-mptl-1 confers MPTL-resistance in T. circumcincta, as in Haemonchus contortus, but that different mutations in Tci-mptl-1 can confer resistance in different populations., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform-based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites' ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.

The nematode genus Bidigiticauda has 2 species ( Bidigiticauda vivipara and Bidigiticauda embryophilum ), which are parasites of bats from the Neotropical region. The present paper describes a new species of Bidigiticauda from a male Artibeus planirostris specimen collected in the Pratigi Environmental Protection Area in Bahia state, Brazil. The new species, Bidigiticauda serrafreirei n. sp., differs from B. embryophilum by having longer spicules, rays 5 and 6 arising from a common trunk and bifurcating in its first third, rays 3 and 4 emerging slightly separated from each other, and dorsal rays reaching the margin of the caudal bursa. The new species also differs from B. vivipara by the dorsal ray bifurcating at the extremity of the trunk. A molecular phylogenetic analysis was conducted to determine the evolutionary affinities of Bidigiticauda serrafreirei n. sp. within the Strongylida, which identified a clade that grouped Bidigiticauda with the other members of the Anoplostrongylinae. However, the molineid subfamilies did not group together, indicating that the family Molineidae is polyphyletic. Further analyses, which include additional taxa and genetic markers, should elucidate the complex relationships within the Molineidae, in particular its subfamilies and the evolution of the traits that define these groups.

Despite the economic, social and ecological importance of the ostertagiine abomasal nematode Marshallagia marshalli, little is known about its life history traits and its adaptations to cope with environmental extremes. Conserved species-specific traits can act as exaptations that may enhance parasite fitness in changing environments. Using a series of experiments, we revealed several unique adaptations of the free-living stages of M. marshalli that differ from other ostertagiines. Eggs were isolated from the feces of bighorn sheep (Ovis canadensis) from the Canadian Rocky Mountains and were cultured at different temperatures and with different media. Hatching occurred primarily as L1s in an advanced stage of development, morphologically very similar to a L2. When cultured at 20 °C, however, 2.86% of eggs hatched as L3, with this phenomenon being significantly more common at higher temperatures, peaking at 30 °C with 28.95% of eggs hatching as L3s. After hatching, free-living larvae of M. marshalli did not feed nor grow as they matured from L1 to infective L3. These life history traits seem to be adaptations to cope with the extreme environmental conditions that Marshallagia faces across its extensive latitudinal distribution in North America and Eurasia. In order to refine the predictions of parasite dynamics under scenarios of a changing climate, basic life history traits and temperature-dependent phenotypic behaviour should be incorporated into models for parasite biology., (Copyright © 2019 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Little is known about the diversity of many parasites infecting camels, with most relying on morphological parameters. DNA extracted from different tissues (n = 90) and fecal samples (n = 101) from dromedary camels (Camelus dromedarius) in Egypt were screened for multiple parasites using different molecular markers. Screening of tissue samples (heart) for Toxoplasma gondii and Sarcocystis spp. was performed using B1 and 18S rRNA gene markers, respectively. T. gondii was further genotyped using multiplex multilocus nested PCR-RFLP (Mn-PCR-RFLP). Sarcocystis was analyzed using PCR-RFLP characterization (XbaI and MboI restriction enzymes). A taxonomically challenging but important group of nematodes (Trichostrongylidae family) were screened using the ITS-2 ribosomal DNA (rDNA) species-specific markers. Furthermore, nested PCR was used for the detection of Cryptosporidium spp. (SSU rRNA gene) and positive samples were genotyped after RFLP (SspI and VspI) and sequencing. Cryptosporidium parvum isolates were subtyped by sequence analysis of the 60-kDa glycoprotein gene. This study revealed that many parasites infect the investigated camels, including T. gondii (1.1%), Sarcocystis spp. (64.4%), Cryptosporidium spp. (5.9%) and Trichostrongylidae nematodes (22.7%). The species contribution for nematodes was as follows: Haemonchus spp. (95.6%), Trichostrongylus axei (26%), Trichostrongylus colubriformis (65.2%) and Cooperia oncophora (60.8%). Mn-PCR-RFLP typing for Toxoplasma was only successful for three markers: 5'-SAG2 (type II), 3'-SAG2 (type II) and alt. SAG2 (type II). PCR-RFLP using XbaI showed possible mixed Sarcocystis infection. Moreover, the Cryptosporidium genotypes detected were C. parvum (IIdA19G1 and IIaA15G1R1), Cryptosporidium rat genotype IV and a novel genotype (camel genotype). This approach revealed the unique Cryptosporidium genotypes infecting the investigated camels, and the high genetic diversity of the investigated parasites., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Trichostrongylid nematodes are a common cause of gastroenteritis in sheep. Despite its worldwide distribution, Teladorsagia circumcincta has not been included in reports listing the various trichostrongyles infecting sheep from Egypt. Herein, we describe the presence of 2 T. circumcincta haplotypes infecting small ruminants from Egypt. For this study, fresh fecal samples were collected from 340 sheep and 115 goats reared at 5 districts in Dakahlia governorate and its surroundings, Egypt. Trichostrongyle eggs were harvested from the samples, and then subjected to DNA isolation and analysis. Polymerase chain reaction (PCR) amplification was carried out for the second internal transcribed spacer of ribosomal DNA ( ITS2 rDNA ). Purified PCR products of T. circumcincta were sequenced, and the revealed sequences were subjected to the nucleotide and phylogenetic analysis. A relatively high prevalence of trichostrongyles eggs was identified in sheep (33.2%) and a lower prevalence was found in goats (14.7%). Molecular analysis revealed, for the first time, 2 sheep herds from Egypt that were infected with T. circumcincta . Both infected herds were raised by the Bedouins in rural areas of El Mahalla El Kubra city. No T. circumcincta infections were found in any of the goats. Nucleotide sequence analysis revealed 2 haplotypes (Te1 and Te2) from 7 successfully sequenced samples (5 from the first and 2 from the second herd). Te1 was the major haplotype in both herds, and Te2 was retrieved from a single sample. Phylogenetic analysis displayed that the Te1 haplotype clustered with one from Cyprus, which might have been introduced to Egypt via goats imported from Cyprus due to a program to improve meat and milk production in Egypt. The present results could be beneficial in understanding the epidemiology of T. circumcincta and other trichostrongyles in Egypt, and have implementations in the effective control strategies used in this region.

The naturalization of alien Reeves's muntjacs (Muntiacus reevesi) on Izu-Oshima Island, Tokyo, Japan, has proceeded intensively over the last five decades. To clarify whether the gastrointestinal helminths of these animals were brought from their original endemic area or were newly acquired in Japan, 32 Reeves's muntjacs trapped on the island were parasitologically examined. In addition to Gongylonema pulchrum in the oesophagus (34.4% prevalence), Chabaudstrongylus ninhae (Dróżdż, 1967) (Trichostrongylidae: Cooperiinae) and Oesophagostomum muntiacum Jian, 1989 (Chabertiidae: Oesophagostominae) were prevalent in the small (28.1%) and large (46.9%) intestines, respectively. For the first time, these trichostrongylid or chabertiid worms were genetically characterized based on partial nucleotide sequences of the nuclear ribosomal RNA gene (rDNA) and mitochondrial DNA cytochrome c oxidase subunit 1 gene (cox-1), and the phylogenetic relationships with other members of their family were explored. Since these two intestinal nematode species are inherent in muntjacs, this study demonstrates a new distribution of exotic helminth species in Japan in accordance with the naturalization of alien mammalian hosts. The molecular genetic data collected here could assist the taxonomic assessment of morphological variants in different Muntiacus spp. and/or of different geographical origins. Furthermore, our data may help to define the phylogenetic relationships among such isolates.

Ashworthius sidemi is a blood-sucking nematode, which has spread out among wild ruminants in several European countries during last decades. The nematode has recently been detected in cattle as well. The distribution of A. sidemi in Russia has not been sufficiently clarified yet. In European part of Russia A. sidemi was formerly registered in sika deer (Cervus nippon) and maral (Cervus elaphus sibiricus) introduced from Asia, and also in aboriginal elks (Alces alces). Taking into consideration the presence of other species of ruminants susceptible to A. sidemi in European Russia, it is necessary to control the spread of this parasite. The specimens of males and females of A. sidemi were found during the autopsies of three naturally infected roe deer (Capreolus capreolus) from Voronezh and Tver regions (European Russia). The species affiliation of the discovered nematodes was determined according to morphological features and confirmed using molecular techniques. The intensity of infection with A. sidemi in two roe deer from Voronezh was 11 and 63 nematodes, and it was 17 nematodes in roe deer from Tver. All of the discovered specimens of A. sidemi were referred to juvenile forms based on features of male bursa morphology and weak development of female reproductive system. In Russia, A. sidemi has not previously been detected in C. capreolus and the present report constitutes the first record of the parasite occurrence in this species of ruminant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ovine parasitic gastroenteritis is a complex disease routinely treated using anthelmintics. Although many different strongyle species may contribute to parasitic gastroenteritis, not all are equally pathogenic: in temperate regions, the primary pathogen is Teladorsagia circumcincta. In this study we investigated benzimidazole and ivermectin resistance on a commercial sheep farm in southeast Scotland. We assessed the impact of species diversity on the diagnosis of resistance using the faecal egg count reduction test and in vitro bioassays, and correlated the results with the frequency of benzimidazole resistance-associated genotypes measured in the T. circumcincta population by pyrosequencing of the β-tubulin isotype-1 gene. Faecal egg count reduction test results showed efficacies of 65% for albendazole and 77% for ivermectin, indicating moderate resistance levels on the farm. However, PCR speciation of the same populations pre- and post-treatment revealed that removal of susceptible species had masked the presence of a highly resistant population of T. circumcincta. Less than 25% of individuals in the pre-treatment populations were T. circumcincta, the remainder consisting of Cooperia curticei, Chabertia ovina, Oesophagostomum venulosum and Trichostrongylus spp. In contrast, post-treatment with albendazole or ivermectin, the majority (88% and 100% respectively) of the populations consisted of T. circumcincta. The egg hatch test for benzimidazole resistance and the larval development test for ivermectin resistance were carried out using eggs obtained from the same populations and the results were broadly consistent with the faecal egg count reduction test. Thirty individual T. circumcincta from each sampling time point were assessed for benzimidazole resistance by pyrosequencing, revealing a high frequency and diversity of resistance-associated mutations, including within the population sampled post-ivermectin treatment. These results highlight the potential diversity of parasite species present on UK farms, and their importance in the diagnosis of anthelmintic resistance. On this particular farm, we demonstrate the presence of a highly dual-resistant population of T. circumcincta, which was strongly selected by treatment with either benzimidazoles or ivermectin, while other potentially less pathogenic species were removed., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The genus Cooperia includes important parasites of ruminants and currently contains 34 accepted species. However, even for those species infecting livestock, there is a considerable lack of molecular information and many species are only identifiable using subtle morphological traits. The present study aimed to provide molecular data to allow diagnosis of Cooperia species infecting cattle. Partial sequences of two mitochondrial (cytochrome oxidase 2, 12S rRNA gene) and two nuclear genes (isotype 1 β tubulin gene including two introns, internal transcribed spacers (ITS) were obtained from morphologically identified specimens of Cooperia pectinata, Cooperia punctata and Cooperia spatulata as well as from larvae of pure Cooperia oncophora and C. punctata laboratory isolates. Pairwise identity of ITS-2 sequences was very high and it was the only region able to identify a specimen as Cooperia sp. However, the ITS-2 was unreliable for diagnosis at the species level. All other marker sequences could not unequivocally be allocated to the genus Cooperia but allowed clear species identification with the exception of the pair C. punctata/C. spatulata for which no significant differences were found for any marker sequence. Maximum-likelihood phylogenetic analyses of individual genes as well as a multi-locus analysis covering all four sequences confirmed that specimen identified as C. spatulata were randomly distributed throughout the C. punctata cluster and formed no group of their own. In contrast, the other Cooperia species formed clearly separated and statistically supported clusters. These data indicate that C. spatulata is most likely only a morphotype of C. punctata and the name should be considered a synonym. Combinations of nuclear and mitochondrial markers should be used to identify morphotypes or cryptic species to benefit from excellent barcoding properties of the latter but allowing proper phylogenetic analyses and controlling for lineage sorting that might occur for mitochondrial genotypes within a species., Competing Interests: The authors have declared that no competing interests exist.

Background: Many trichostrongylid nematode species are reported to infect bison, some of which are major causes of disase and production loss in North American bison herds. However, there is little information on the species distribution and relative abundance of these parasites in either commercial or conservation herds. This is largely because trichostrongylid nematode species cannot be distinguished by visual microscopic examination of eggs present in feces. Consequently, we have applied ITS2 rDNA nemabiome metabarcoding to describe the trichostrongyle parasite species diversity in 58 bison production groups derived from 38 commercial North American plains bison (Bison bison bison) herds from across western Canada, and two bison conservation herds located in Elk Island National Park (EINP) [plains bison and wood bison (Bison bison athabascae)] and one in Grasslands National Park (GNP) (plains bison)., Results: We report much higher infection intensities and parasite species diversity in commercial bison herds than previously reported in beef cattle herds grazing similar latitudes. Predominant trichostrongyle parasite species in western Canadian commercial bison herds are those commonly associated with Canadian cattle, with Ostertagia ostertagi being the most abundant followed by Cooperia oncophora. Combined with high fecal egg counts in many herds, this is consistent with significant clinical and production-limiting gastrointestinal parasitism in western Canadian bison herds. However, Haemonchus placei was the most abundant species in five of the production groups. This is both surprising and important, as this highly pathogenic blood-feeding parasite has not been reported at such abundance, in any livestock species, at such northerly latitudes. The presence of Trichostrongylus axei as the most abundant parasite in four herds is also unusual, relative to cattle. There were striking differences in parasite communities between the EINP and commercial bison herds. Most notably, Orloffia bisonis was the predominant species in the wood bison herd despite being found at only low levels in all other herds surveyed., Conclusions: This study represents the most comprehensive description of parasite communities in North American bison to date and illustrates the power of deep amplicon sequencing as a tool to study species diversity in gastrointestinal nematode communities.

Background: Libyostrongylus douglassii, Libyostrongylus dentatus and Libyostrongylus magnus are nematodes that infect ostriches. The first species has been identified in ostriches from Africa, Europe, Americas and Oceania. Although the natural range of ostriches is Africa, L. dentatus was first described in birds from the USA and later identified in Brazil, where co-infections with L. douglassii have been commonly reported. Libyostrongylus magnus is known from the original description only. There are a few reports on infections with L. douglassii in ostriches from Africa and all farmed birds examined are from the southern region of the continent. The aim of this report was to verify Libyostrongylus spp. infections in wild ostriches from Ethiopia. Fecal samples from ostriches, Struthio molybdophanes, were collected and submitted to coproculture. Infective larvae were identified to the species level based on general morphology and morphometry. In addition, phylogenetic analysis of the first and second internal transcribed spacer (ITS1 and ITS2) of the nuclear ribosomal DNA was performed., Results: Infective larvae from Ethiopian ostriches had the morphological characteristics of L. dentatus. Confidence interval estimate for sheath tail length from Ethiopian Libyostrongylus sp. isolates overlapped one for Brazilian L. dentatus. Neighbor-joining and Maximum Likelihood phylogenetic trees based on sequences of the ITS1 and ITS2 regions revealed that the Ethiopian samples belong to the L. dentatus species clade. Monospecific infections with L. dentatus were confirmed in Ethiopian wild ostriches, opposed to the co-infections typically found in the Americas., Conclusions: To our knowledge, this is the first record of L. dentatus from African ostriches, the region from which this parasite originated.

Marshallagia marshalli (Nematoda: Trichostrongylidae) infection can lead to serious parasitic gastroenteritis in sheep, goat, and wild ruminant, causing significant socioeconomic losses worldwide. Up to now, the study concerning the molecular biology of M. marshalli is limited. Herein, we sequenced the complete mitochondrial (mt) genome of M. marshalli and examined its phylogenetic relationship with selected members of the superfamily Trichostrongyloidea using Bayesian inference (BI) based on concatenated mt amino acid sequence datasets. The complete mt genome sequence of M. marshalli is 13,891 bp, including 12 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. All protein-coding genes are transcribed in the same direction. Phylogenetic analyses based on concatenated amino acid sequences of the 12 protein-coding genes supported the monophylies of the families Haemonchidae, Molineidae, and Dictyocaulidae with strong statistical support, but rejected the monophyly of the family Trichostrongylidae. The determination of the complete mt genome sequence of M. marshalli provides novel genetic markers for studying the systematics, population genetics, and molecular epidemiology of M. marshalli and its congeners.

The history of European bison Bison bonasus Linnaeus, 1758 has been stormy since its extinction in the wild after the First World War. Due to the fact that the species was restored from just 12 founders, further expansion has suffered from low genetic variability, rendering the bison vulnerable to various pathogens due to inbreeding depression. Parasites are recognised as a key biological threat to bison population. Thus, parasitological examination including monitoring of the level of anthelmintic resistance in a herd should be a routine procedure involved in management and protection of European bison. This study was conducted in a group of 27 bison kept in a European bison breeding centre in Sweden. In April 2015, a faecal egg count reduction test (FECRT) was performed in animals with ≥ 100 gastrointestinal nematode (GIN) eggs per gram faeces, to determine effectiveness of fenbendazole (FBZ) treatment. Additionally, the third stage larvae were cultured for molecular examination by a conventional PCR as well as by real-time quantitative PCR (q-PCR) for detection of the blood-sucking nematode Haemonchus contortus. Faecal sampling was conducted 1 day before and 8 days after deworming each animal. Anthelmintic treatment turned to be entirely efficient toward intestinal nematodes of genera Nematodirus and Trichuris, whereas shedding of strongylid eggs from the subfamily Ostertagiinae was reduced from 81 to 30%. Polymerase chain reaction (PCR) on cultured third-stage larvae (L3) before treatment was positive for H. contortus, Ostertagia ostertagi and Cooperia oncophora, whereas post-treatment examination revealed exclusively the DNA of H. contortus. Thus, only H. contortus was involved in post-treatment faecal egg count (FEC). FECRT showed that the reduction in strongylid FEC to FBZ in the examined bison herd was 87% (95%-confidence intervals [95% CI] = 76-93), suggesting reduced efficacy of FBZ to strongylid GIN including mainly H. contortus.

Maternal dietary protein deficiency and gastrointestinal nematode infection during early pregnancy have negative impacts on both maternal placental gene expression and fetal growth in the mouse. Here we used next-generation RNA sequencing to test our hypothesis that maternal protein deficiency and/or nematode infection also alter the expression of genes in the developing fetal brain. Outbred pregnant CD1 mice were used in a 2×2 design with two levels of dietary protein (24% versus 6%) and two levels of infection (repeated sham versus Heligmosomoides bakeri beginning at gestation day 5). Pregnant dams were euthanized on gestation day 18 to harvest the whole fetal brain. Four fetal brains from each treatment group were analyzed using RNA Hi-Seq sequencing and the differential expression of genes was determined by the edgeR package using NetworkAnalyst. In response to maternal H. bakeri infection, 96 genes (88 up-regulated and eight down-regulated) were differentially expressed in the fetal brain. Differentially expressed genes were involved in metabolic processes, developmental processes and the immune system according to the PANTHER classification system. Among the important biological functions identified, several up-regulated genes have known neurological functions including neuro-development (Gdf15, Ing4), neural differentiation (miRNA let-7), synaptic plasticity (via suppression of NF-κβ), neuro-inflammation (S100A8, S100A9) and glucose metabolism (Tnnt1, Atf3). However, in response to maternal protein deficiency, brain-specific serine protease (Prss22) was the only up-regulated gene and only one gene (Dynlt1a) responded to the interaction of maternal nematode infection and protein deficiency. In conclusion, maternal exposure to GI nematode infection from day 5 to 18 of pregnancy may influence developmental programming of the fetal brain., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The protective capacities of a native double-domain activation-associated secreted protein (ndd-ASP)-based vaccine against the cattle intestinal nematode Cooperia oncophora has previously been demonstrated. However, protection analysis upon vaccination with a recombinantly produced antigen has never been performed. Therefore, the aim of the current study was to test the protective potential of a Pichia-produced double-domain ASP (pdd-ASP)-based vaccine against C. oncophora. Additionally, we aimed to compare the cellular and humoral mechanisms underlying the vaccine-induced responses by the native (ndd-ASP) and recombinant vaccines. Immunisation of cattle with the native C. oncophora vaccine conferred significant levels of protection after an experimental challenge infection, whereas the recombinant vaccine did not. Moreover, vaccination with ndd-ASP resulted in a higher proliferation of CD4-T cells both systemically and in the small intestinal mucosa when compared with animals vaccinated with the recombinant antigen. In terms of humoral response, although both native and recombinant vaccines induced similar levels of antibodies, animals vaccinated with the native vaccine were able to raise antibodies with greater specificity towards ndd-ASP in comparison with antibodies raised by vaccination with the recombinant vaccine, suggesting a differential immune recognition towards the ndd-ASP and pdd-ASP. Finally, the observation that animals displaying antibodies with higher percentages of recognition towards ndd-ASP also exhibited the lowest egg counts suggests a potential relationship between antibody specificity and protection., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A 1023 bp full length cDNA encoding Teladorsagia circumcincta GAPDH (TeciGAPDH) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth GAPDH sequences. The predicted protein consisted of 341 amino acids and was present as a single band of about 38 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciGAPDH with homologues from other helminths showed that the greatest similarity (93%) to the GAPDH of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 °C, the optimum pH for TeciGAPDH activity was pH 8, the V max was 1052 ± 23 nmol min -1  mg -1 protein and the apparent K m for the substrate glyceraldehyde-3-phosphate was 0.02 ± 0.01 mM (both mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TeciGAPDH in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native GAPDH indicates similar antigenicity of the two proteins., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Haemonchus contortus is a major parasite of small ruminants and its blood-feeding behaviour causes effects ranging from mild anaemia to death. Knowledge of the genetic variation within and among H. contortus populations can provide the foundation for understanding transmission patterns and aid in the control of haemonchosis. Adult male H. contortus were collected from three geographical regions in Egypt. The second internal transcribed spacer (ITS2) of nuclear ribosomal DNA was amplified using the polymerase chain reaction (PCR) and sequenced directly. The population genetic diversity and sequence variations were determined. Nucleotide sequence analyses revealed one genotype (ITS2) in all worms, without genetic differentiation. The similarity in population genetic diversity and genetic patterns observed among the three geographical regions could be attributed to possible movement between the sites. This is the first study of genetic variation in H. contortus in Egypt. The present results could have implications for the rapid characterization of H. contortus and other trichostrongyloid nematodes, and evaluation of the epidemiology of H. contortus in Egypt.

Metazoan parasites have to survive in many different niches in order to complete their life-cycles. In the absence of reliable methods to manipulate parasite genomes and/or proteomes, identification of the molecules critical for parasite survival within these niches has largely depended on comparative transcriptomic and proteomic analyses of different developmental stages of the parasite; however, changes may reflect differences associated with transition between developmental stages rather than specific adaptations to a particular niche. In this study, we compared the transcriptome of two fourth-stage larval populations of the nematode parasite, Teladorsagia circumcincta, which were of the same developmental stage but differed in their location within the abomasum, being either mucosal-dwelling (MD) or lumen-dwelling (LD). Using RNAseq, we identified 57 transcripts which were significantly differentially expressed between MD and LD larvae. Of these transcripts, the majority (54/57) were up-regulated in MD larvae, one of which encoded for an ShKT-domain containing protein, Tck6, capable of modulating ovine T cell cytokine responses. Other differentially expressed transcripts included homologues of ASP-like proteins, proteases, or excretory-secretory proteins of unknown function. Our study demonstrates the utility of niche- rather than stage-specific analysis of parasite transcriptomes to identify parasite molecules of potential importance for survival within the host.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

The present study was performed to analyze molecularly the phylogenetic positions of human-infecting Trichostrongylus species in Mazandaran Province, Iran, which is an endemic area for trichostrongyliasis. DNA from 7 Trichostrongylus infected stool samples were extracted by using in-house (IH) method. PCR amplification of ITS2-rDNA region was performed, and products were sequenced. Phylogenetic analysis of the nucleotide sequence data was performed using MEGA 5.0 software. Six out of 7 isolates had high similarity with Trichostrongylus colubriformis , while the other one showed high homology with Trichostrongylus axei registered in GenBank reference sequences. Intra-specific variations within isolates of T. colubriformis and T. axei amounted to 0-1.8% and 0-0.6%, respectively. Trichostrongylus species obtained in the present study were in a cluster with the relevant reference sequences from previous studies. BLAST analysis indicated that there was 100% homology among all 6 ITS2 sequences of T. colubriformis in the present study and most previously registered sequences of T. colubriformis from human, sheep, and goat isolates from Iran and also human isolates from Laos, Thailand, and France. The ITS2 sequence of T. axei exhibited 99.4% homology with the human isolate of T. axei from Thailand, sheep isolates from New Zealand and Iran, and cattle isolate from USA.

Background: Traditional methods of detecting and identifying gastrointestinal nematode infections in small ruminants, including sheep and goats, are time-consuming and lack in sensitivity and specificity. Recently, we developed an automated multiplexed-tandem (MT)-PCR platform for the diagnosis and identification patent infections with key genera/species of gastrointestinal nematodes of sheep and validated this approach in detailed experiments carried out in Australia. In the present study, we deployed this diagnostic platform in Scotland and Belgium to test samples from naturally infected sheep in these environments and to validate the MT-PCR platform relative to traditional diagnostic methods routinely used by local laboratories., Results: MT-PCR detected all microscopy positive samples and there was a significant agreement between the results of the different test methods in terms of the species detected and their relative proportion in a test sample, however, for some samples, there were discrepancies between the results of the different test methods. Selective sequencing of purified MT-PCR products demonstrated the results to be 100% specific., Conclusions: The MT-PCR platform is an advanced method for the species/genus-specific diagnosis of gastrointestinal nematode infections in small ruminants and has demonstrated utility when deployed in different countries and climatic zones. The platform is user-friendly due to the largely automated procedure and has high versatility in that it can achieve a specific diagnosis from different types of sample templates, including larval culture and faecal samples. With appropriate modifications of the primers used, the MT-PCR platform also provides potential for the diagnosis of a variety of other pathogens of veterinary or medical importance.

Among gastro-intestinal nematodes, the blood-sucking worms belonging to the subfamily of Haemonchinae are considered to be of pathogenic and economic great importance, particularly in small ruminants. Haemonchus contortus, primary found in domestic ruminants and wild bovines (Mouflon, Chamois), is probably the most studied, but occurrence of Ashworthius sidemi has gradually increased over recent years, especially in Cervids and free roaming wild bovid as the European bison in eastern Europe, and some cases of co-infestation were recently observed on five Roe deer (Capreolus capreolus) and one Red deer (Cervus elaphus) in France. If the diagnosis is possible on the morphological features for adult worms for helminthologists, the identification on some stages (female, subadult, eggs and larvae) is difficult or impossible. Sequencing ND4 domain from the mitochondrial DNA of H. contortus and A. sidemi worms, we observed clearly two distinct clades, with an inter-specific divergence of 28.1%. Basing on this specific domain, a multiplex PCR-based method was developed: new primers were designed and used pooled in one mix PCR, producing amplicons of 454bp for H. contortus and 330bp for A. sidemi, allowing a trivial and an inexpensive taxonomic affiliation after migration. This multiplex PCR-based method was developed here to distinguish H. contortus and A. sidemi regardless their developmental stage, easy to use for highlighting co-infestation cases in both wild and domestic ruminants. It is a non-invasive approach appearing as a good diagnostic tool relevant to coprological cultures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Resistance to benzimidazoles (BZs) in trichostrongyloid nematodes is a worldwide problem for livestock production, particularly regarding small ruminants. Sensitive and reliable methods are required to assess anthelmintic resistance status. Currently available methods for BZ resistance detection can be divided into three main groups, in vivo (e.g. faecal egg count reduction test), in vitro (e.g. egg hatch assay) and molecular tests. Three single nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene of various nematode species correlate with BZ resistance. While PCR-based methods have been reported for the three most economically important nematodes of sheep, namely, Trichostrongylus, Haemonchus and Teladorsagia, pyrosequencing assays are so far only available for the latter two. Here, the design and evaluation of pyrosequencing assays for isotype-1 and isotype-2 β-tubulin genes of Trichostrongylus colubriformis are described. PCR fragments carrying the susceptible and corresponding resistant genotype were combined in defined ratios to evaluate assay sensitivity and linearity. The correlation between the given and the measured allele frequencies of the respective SNPs (codons F167Y, E198A and F200Y) was very high. Pyrosequencing assays for Haemonchus, Teladorsagia and Trichostrongylus were subsequently used for a BZ resistance survey, carried out in the three European countries, namely Ireland, Italy and Switzerland. Larval cultures obtained from field survey samples in 2012 and 2013 were used for pyrosequencing. The test was applied when the target species represented at least 10% of the sample. Trichostrongylus and Teladorsagia were detected in all countries' samples whereas Haemonchus was not detected in samples from Ireland. SNPs in isotype-1 associated with resistance were detected for all three species, with frequencies at codon F200Y far exceeding those at codons F167Y and E198A. Elevated SNP frequencies in isotype-2 of Trichostrongylus were only rarely detected. Farms with BZ resistance-associated SNP frequencies above 10% were most often found in Switzerland followed by Ireland and Italy., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The efficacy of ivermectin (IVM) against gastrointestinal nematodes in Danish cattle was assessed by faecal egg count reduction test (FECRT). Six cattle farms with history of clinical parasitism and avermectin use were included. On the day of treatment (Day 0), 20 naturally infected calves per farm (total n = 120) were stratified by initial faecal egg counts (FEC) and randomly allocated to a treatment group dosed with 0.2 mg IVM kg -1 body weight s.c. (IVM; n = 10) or an untreated control group (CTL; n = 10). Individual FEC were obtained at Day 0 and Day 14 post-treatment and pooled faeces by group were cultured to isolate L3 for detection of Ostertagia ostertagi and Cooperia oncophora by qPCR. Treatment efficacies were analysed using the recommended WAAVP method and two open-source statistical procedures based on Bayesian modelling: 'eggCounts' and 'Bayescount'. A simulation study evaluated the performance of the different procedures to correctly identify FEC reduction percentages of simulated bovine FEC data representing the observed real data. In the FECRT, reduced IVM efficacy was detected in three farms by all procedures using data from treated animals only, and in one farm according to the procedures including data from treated and untreated cattle. Post-treatment, O. ostertagi and C. oncophora L3 were detected by qPCR in faeces of treated animals from one and three herds with declared reduced IVM efficacy, respectively. Based on the simulation study, all methods showed a reduced performance when FEC aggregation increased post-treatment and suggested that a treatment group of 10 animals is insufficient for the FECRT in cattle. This is the first report of reduced anthelmintic efficacy in Danish cattle and warrants the implementation of larger surveys. Advantages and caveats regarding the use of Bayesian modelling and the relevance of including untreated cattle in the FECRT are discussed., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This paper focuses on the species diversity among the Trichostrongylidae Leiper, 1912 (Nematoda: Strongylida), and complexity of the family systematics. Polymorphism (subfamilies: Ostertagiinae, Cooperiinae and Haemonchinae), the presence of cryptic species (genus: Teladorsagia) and hybridization (genera: Cooperia, Haemonchus and Ostertagia) are presented and discussed, considering both morphological and molecular evidence. Some of these phenomena are common, nevertheless not sufficiently understood, which indicates the need for expanding the current state of knowledge thereof. Within the Trichostrongylidae, species distinction supported merely by morphological features is difficult, and requires confirmation by means of molecular methods. The parasitic nematode taxonomy is complicated mainly by the genus Teladorsagia, but complexity may also be expected among other Ostertagiinae (e.g. in the genera Ostertagia and Marshallagia). The data presented here show that the members of the Trichostrongylidae can significantly complicate unambiguous species identification. Hence, it is essential to consider the phenomena mentioned, to gather valid and comparable data on the biodiversity of this family.

In reproducing ewes, a periparturient breakdown of immunity is often observed to result in increased fecal egg excretion, making them the main source of infection for their immunologically naive lambs. In this study, we expanded a simulation model previously developed for growing lambs to explore the impact of the genotype (performance and resistance traits) and host nutrition on the performance and parasitism of both growing lambs and reproducing ewes naturally infected with Teladorsagia circumcincta. Our model accounted for nutrient-demanding phases, such as gestation and lactation, and included a supplementary module to manage the age structure of the ewe flock. The model was validated by comparison with published data. Because model parameters were unknown or poorly estimated, detailed sensitivity analysis of the model was performed for the sheep mortality and the level of infection, following a preliminary screening step. The parameters with the greatest effect on parasite-related outputs were those driving animal growth and milk yield. Our model enables different parasite-control strategies (host nutrition, breeding for resistance and anthelmintic treatments) to be assessed on the long term in a sheep flock. To optimize in silico exploration, the parameters highlighted by the sensitivity analysis should be refined with real data.

Background: Refugia based anthelmintic protocols aim to reduce the rate of development of anthelmintic resistance in gastrointestinal nematodes (GIN). Previous studies have illustrated the impact of different drenching regimes on drug efficacy and animal growth; however, the impact on nematode populations has yet to be characterised within natural infections. This study investigated the changes in species composition of GIN throughout the grazing season, following implementation of four different ivermectin drenching regimes over six years: neo-suppressive monthly treatment (NST), targeted selective treatment (TST), strategic prophylactic treatment (SPT) and treatment upon observation of clinical signs (MT)., Methods: Lambs were grazed on one of eight replicate paddocks each grazing season following treatment regimes assigned in year 1. Faecal samples were collected fortnightly from all animals and hatched to first stage larvae (L1). DNA was extracted from individual L1 and a multiplex PCR assay targeting the internal transcribed spacer 2 (ITS2) region of Teladorsagia circumcincta, Trichostrongylus spp. and Haemonchus contortus conducted. Other species were identified using species-specific PCR. Worm-naïve tracer lambs were grazed on the paddocks at the start and end of each grazing season and adult worms recovered at post mortem to investigate the parasite population on pasture., Results: Results showed an overall decrease in species diversity in egg output from the NST group which occurred within a single grazing season and was consistent throughout the experiment. Species diversity was protected over six years in groups implementing TST, SPT and MT treatment regimes, designed to offer refugia. The expected shift in species prevalence throughout the season from Teladorsagia to Trichostrongylus was observed in all but the NST group where only Teladorsagia spp. were recovered from trial lambs by the end of the experiment. Worm burdens indicated the presence of several species at relatively low abundance on pasture in the NST group in 2011. However, these species were not represented in egg output from trial lambs, probably due to the frequent anthelmintic treatment administered throughout the grazing season., Conclusion: The molecular methods utilised here worked well. The comparable results of the three refugia-based treatment regimes suggest that nematode diversity can be maintained using part or whole group treatments if a rich supra-population of parasites are available to re-infect animals post treatment.

Gastrointestinal nematodes within the subfamily Ostertagiinae (Teladorsagia, Ostertagia, and Marshallagia et al.) are among the most common infections of domesticated livestock. These parasites are of particular interest, as many of the species within this group are of economic importance worldwide. Traditionally, nematode species designations have been based on morphological criteria. However, this group possesses poorly defined species. There is an urgent need to develop a reliable technique that can distinguish species of Ostertagiinae. DNA barcoding has been proved to be a powerful tool to identify species of birds, mammals, and arthropods, but this technique has not yet been examined for identifying species of Ostertagiinae. In this study, a total of 138 mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) sequences from individuals representing 11 species of Ostertagiinae were acquired by PCR for the first time. The specimens were collected from pastoral area of northern China. Genetic divergence analyses showed that mean interspecific Kimura two-parameter distances of COI (13.61 %) were about four times higher than the mean value of the intraspecific divergence (3.69 %). Then, the performance of the COI to identify species of Ostertagiinae was evaluated by identification success rates using nearest neighbor (NN) and BLASTn. The results indicated that the rates of correct sequence identification for COI were high (>80 %) when using the NN and BLASTn methods. Besides, the deep lineage divergences are detected in Teladorsagia circumcincta. Meanwhile, the analyses also detected no genetic differentiation between some species such as Ostertagia hahurica and Ostertagia buriatica. These results indicate that the traditional status of species within Ostertagiinae should be closely examined based on the molecular data.